TFIIS enhances transcriptional elongation through an artificial arrest site in vivo - PubMed (original) (raw)

TFIIS enhances transcriptional elongation through an artificial arrest site in vivo

D Kulish et al. Mol Cell Biol. 2001 Jul.

Abstract

Transcriptional elongation by RNA polymerase II has been well studied in vitro, but understanding of this process in vivo has been limited by the lack of a direct and specific assay. Here, we designed a specific assay for transcriptional elongation in vivo that involves an artificial arrest (ARTAR) site designed from a thermodynamic theory of DNA-dependent transcriptional arrest in vitro. Transcriptional analysis and chromatin immunoprecipitation experiments indicate that the ARTAR site can arrest Pol II in vivo at a position far from the promoter. TFIIS can counteract this arrest, thereby demonstrating that it possesses transcriptional antiarrest activity in vivo. Unexpectedly, the ARTAR site does not function under conditions of high transcriptional activation unless cells are exposed to conditions (6-azauracil or reduced temperature) that are presumed to affect elongation in vivo. Conversely, TFIIS affects gene expression under conditions of high, but not low, transcriptional activation. Our results provide physical evidence for the discontinuity of transcription elongation in vivo, and they suggest that the functional importance of transcriptional arrest sites and TFIIS is strongly influenced by the level of transcriptional activation.

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Figures

FIG. 1

FIG. 1

Rationale for the artificial arresting DNA sequence design. The modules of the ARTAR site and three elongation complexes (gray boxes) occupying them are shown schematically. There are three similar arresting sections containing a spacer (SPC), a strong stabilizing hybrid (SH), a weak oligo(U) destabilizing hybrid (UH), and a strong stopping downstream bubble-flanking duplex (DBF). C-UH is an oligo(U) destabilizing hybrid that is modified by the clamp-forming element. The nascent transcript (dark grey line), DNA (open box in both duplex and RNA-DNA hybrid forms), active Pol II centers (triangles), and transcription bubble borders (brackets) are indicated. The upper panel shows three elongation complexes stalled by DBFs and prone to backtracking because of the weak UH and SPC. These complexes are shown to isomerize (backtrack) into the arrested conformations shown in the bottom panel that are stabilized by the SHs. The hairpin at the downstream ternary complex is predicted to form because of the C-UH structure.

FIG. 2

FIG. 2

The nucleotide sequence of the ARTAR site. The names of the ARTAR elements correspond to those described in the legend to Fig. 1.

FIG. 3

FIG. 3

Effect of the ARTAR site and TFIIS on lacZ expression. β-Galactosidase levels in the indicated strains (artar designates the mutated U*C derivative) grown in medium containing 2% glucose (A), 2% raffinose (B), and 2% galactose (C). Note the differences in scales for β-galactosidase levels in the various panels. (D) RNA levels in the indicated strains grown in 2% galactose medium.

FIG. 4

FIG. 4

Inverse effects of the ARTAR site and TFIIS as a function of galactose (gal) concentration. β-Galactosidase levels in the indicated strains grown in medium containing 2% raffinose, 0.1% glucose, and the indicated concentrations of galactose. The scale for β-galactosidase levels is exponential.

FIG. 5

FIG. 5

Analysis of Pol II occupancy by chromatin immunoprecipitation. (A) Diagram showing the location of primer pairs for analysis of Pol II occupancy over the lacZ region. (B) Cross-linked chromatin from the indicated HA-Rpb3-containing strains grown in glucose (glu), raffinose (raf), or galactose (gal) medium immunoprecipitated with an HA-specific antibody and analyzed by quantitative PCR primers to the indicated regions.

FIG. 6

FIG. 6

ARTAR and TFIIS dependence of lacZ expression in the presence of the indicated concentrations of 6-AU.

FIG. 7

FIG. 7

ARTAR and TFIIS dependence of the kinetics of lacZ expression after induction with 2 or 0.1% galactose.

FIG. 8

FIG. 8

ARTAR and TFIIS dependence of lacZ expression in cells grown at 16°C.

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